Abstract

BACKGROUND:

West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease.

METHODS:

Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis.

RESULTS:

Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease.

CONCLUSIONS:

Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

Identification of bacterial populations in subgingival plaque of West Virginians. Bacterial composition in plaque samples was determined using 16S rRNA gene sequencing in 2 low and 5 high oral disease COHRA participants, ranked based on periodontal exams, and 5 Cognitive study participants, ranked based on caries index. DM is a low periodontal disease control sample obtained through the West Virginia University Dental Clinic. Each numbered box indicates the percentage of clones of the type-specific bacterial 16S rRNA gene relative to the total number of clones sequenced, which is indicated at the bottom of each column. The color of the box reflects observed counts.

Statistical analyses of bacterial populations in low and high disease plaque. A) Principal coordinate analysis of bacterial communities from subgingival plaque of West Virginians with low oral disease (blue) as compared to plaque of West Virginians with varying degrees of oral disease (red). B) The Unifrac algorithm was used to compute the unique branch length for a given sub-sample. Cluster analysis split low (blue) and diseased samples (red) into different clades. Support for the clusters was evaluated by jackknife tests (1000 replicates). T was the least diseased sample in the Cognitive study, as ranked in Table 2. The 'D' has been removed from sample notations in these figures for clarity.

Comparison of 16S rRNA gene analyses using sequencing and HOMIM. The frequency of bacterial types (by percentage), determined by 16S rRNA sequencing, of 4 plaque samples from low disease and 5 plaque samples from high disease West Virginians, ranked based on criteria defined in Materials and Methods, was compared with the microarray signal intensity obtained from the same samples in HOMIM analyses. Bacterial types above the red line were more frequent in low disease.

Phylogenetic trees of Selenomonas and Veillonella isolated from individual plaque samples. 16S rRNA gene sequences from a single high disease plaque sample (DA, red text) and a single low disease plaque sample (DB, blue text) were used to generate phylogenetic trees of Selenomonas and Veillonella, respectively. The x-axis indicates percent difference in 16S rRNA gene sequence. The separated clusters of Selenomonas show that this population descended from independently colonizing but phylogenetically related bacteria. Veillonella from DB exhibited limited diversity.